1. Field of the Invention
The invention in the fields of microbiology and medicine relates to methods for rapid early detection of mycobacterial disease in humans based on the presence of antibodies to particular “early” mycobacterial antigens which have not been previously recognized for this purpose. Assay of such antibodies on select partially purified or purified mycobacterial preparations containing such early antigens permits diagnosis of TB earlier than has been heretofore possible. Also provided is a surrogate marker for screening populations at risk for TB, in particular subjects infected with human immunodeficiency virus (HIV). The invention is also directed to vaccine compositions and methods useful for preventing or treating TB.
2. Description of the Background Art
The incidence of tuberculosis has shown a rapid increase in recent years, not only in the developing countries, but also in crowded urban settings in the US and in specific subsets of our society, including the homeless, IV drug users, HIV-infected individuals, immigrants and refugees from high prevalence endemic countries (Raviglione, M C et al., 1995. JAMA. 273:220-226). Studies show that these populations are at a significantly greater risk of developing tuberculosis, and also serve as the reservoir of infection for the community as a whole (Raviglione, M C et al., 1992, Bull World Health Organization. 70:515-526; Raviglione, M C et al., 1995. JAMA. 273:220-226). None of the currently used methods for diagnosis of tuberculosis identify individuals with active but sub-clinical infection, and the disease is generally detected when the individuals are already infectious. Design of new diagnostic assays requires knowledge of antigens expressed by the bacteria during their in vivo survival. Most current studies of antigens of Mycobacterium tuberculosis (Mtb); also abbreviated herein are focused on antigens present in the culture filtrates of bacteria replicating actively in vitro, with the presumption that the same molecules are expressed by the in vivo bacteria.
A vast majority of the Mtb infected individuals develop immune responses that arrest progression of infection to clinical TB, and also prevent the latent bacilli from reactivating to cause clinical disease, whereas about 10-15% of the infected individuals progress to developing primary or reactivation TB. Understanding the host-pathogen interactions that occur after infection, but prior to development of clinical TB (pre-clinical TB) is required both for the design of effective vaccines and for development of diagnosis of early disease.
Several studies have shown that Mtb adapts to different environments in broth media    (Garbe, T R et al., 1999, Infect. Immun. 67:460-465; Lee, B-Y et al., 1995, J. Clin. Invest. 96:245-249; Wong, D K et al., 1999, Infect. Immun. 67:327-336) and during intracellular residence by altering its gene expression (8, 22, 34).    Clark-Curtiss, J E et al., 1999, p. 206-210. In Proceedings of Thirty-Fourth Tuberculosis-Leprosy Research Conference, San Francisco, Calif., Jun. 27-30.    Lee et al., supra; Smith, I et al., 1998, Tuber. Lung Dis. 79:91-97). Earlier studies from the present inventors' laboratory with cavitary and non-cavitary TB patients have also shown that the in vivo environment in which the bacilli replicate affects the profile of the antigenic proteins expressed by Mtb (Samanich, K M et al., 1998, J. Infect. Dis. 178:1534-1538; Laal et al., U.S. Pat. No. 6,245,331 (2001)).
One objective of the present invention was to identify the antigens expressed by inhaled Mtb during the pre-clinical stages of TB. There are no markers to identify non-diseased humans with an active infection with Mtb, but the rabbit model of TB closely resembles TB in immuno-competent humans in that both species are outbred, both are relatively resistant to Mtb, and in both the caseous lesions may liquify and form cavities (Converse, P J et al., 1996, Infect. Immun. 64:4776-4787). Studies have shown that on being inhaled, the bacilli are phagocytosed by (non specifically) activated alveolar macrophages (AM) which either destroy or allow them to multiply. If the bacilli multiply, the AM die and the released bacilli are phagocytosed by non activated monocyte/macrophages that emigrate from the bloodstream. Intracellular replication and host cell death continue for 3-5 weeks, when both cellular and humoral immune responses are elicited (Lurie, M B, 1964. Chapter VIII, p. 192-222, In M. B. Lurie (ed.) Resistance to tuberculosis: experimental studies in native and acquired defensive mechanisms. Harvard University Press, Cambridge, Mass.; Lurie, M B et al., 1965, Bact. Rev. 29:466-476; Dannenberg, A M., Jr., 1991, Immunol. Today. 12:228-233). Lymphocytes and macrophages enter the foci of infection, and if they become activated bacillary replication is controlled, if not, the infection progresses to clinical disease. During these initial stages of bacillary replication and immune stimulation, there are no outward signs of disease except the conversion of cutaneous reactivity to PPD. The antigens of Mtb expressed, and their interaction with the immune system during these pre-clinical stages of TB is not delineated.